Identification of bacteria has traditionally been by morphologic features and phenotypic testing. Identification of some common organisms (e.g., Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli) can be rapid (e.g., less than one hour), exploiting their characteristic morphology and a few selected phenotypic tests. However, the identification of most organisms is slow, requiring hours to days for a definitive answer. More recently the use of genomics such as sequencing ribosomal RNA genes or housekeeping genes has proved to be a useful tool. Although sequencing specific genes is a powerful discriminatory tool, the current methodology requires one or more days before a result is available. A logical extension of sequencing genes is to use the gene products for bacterial identification. Significant variations in a structural gene sequence would result in variations in the protein product. In the last 5 years, preliminary work in the analysis of bacterial proteins for identification has been performed using Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-ToF MS) and Surface-Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry (SELDI-ToF MS). Last year we explored using SELDI for identification of selected gram-positive and gram-negative bacteria. This year, with the multi-department Clinical Center purchase of the MALDI-ToF MS system, we have expanded our studies to use this system. Preliminary studies indicate that unique, reproducible profiles could be obtained for Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis. The advantage of the MALDI system is that whole cells could be analyzed, reducing the variability of bacterial lysis which was required with the SELDI system. The difficulty with the MALDI system is the complexity of the profiles. We believe this can be resolved with the recent purchase of software for data analysis.